A fundamental feature of a living system is the integrated network of biochemical pathways and processes that generate a balanced and robust metabolism. The long term goal of the research in my laboratory is to contribute to our understanding of the metabolic network and potential of the system by using a microbial model. This goal requires the identification and characterization of both the cellular components and the connections between them. To accomplish our goal, we utilize focused molecular, and broad systems approaches, the latter consisting of in vivo phenotypic analyses of the system. Over the last 16 years we have developed thiamine biosynthesis in S. enterica as model system to probe metabolic integration, identifying and describing the metabolic network radiating outward from the thiamine biosynthetic pathway. These studies have uncovered an extensive, robust system and highlight the metabolic potential that exists in a cellular network. Work described in this proposal continues these efforts and will contribute to our understanding of the molecular details of thiamine biosynthesis and its integration with other metabolic processes. This will be accomplished by i) defining the molecular mechanism used by the SAM radical enzyme ThiC to generate the pyrimidine moiety of thiamine, ii) characterizing the physiological targets to which ApbC can transfer an Fe-S cluster and dissecting the mechanism involved, and iv) dissecting a recently discovered link between the histidine biosynthetic pathway and the formation of the first precursor to thiamine. The goals of this proposal will be accomplished through the use of modern chemical, biochemical, biophysical, molecular and genetic techniques. Results of the work described here will contribute to our understanding of basic physiology and metabolism by characterizing molecular components and global connections in metabolism that are not yet understood. These efforts will result in the description of the metabolic potential of the system. Understanding the potential inherent in a system allows the productive manipulation, modeling and reconstructions of metabolism which will lead in the future to improvements in the environment, human and animal health.